Lymantria Dispar Mortality in Pupal Stage Caused by Entomophaga Maimaiga in Bulgaria and Serbia

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UDC: 632.78:632.5

Original scientific paper Lymantria dispar Mortality in Pupal Stage Caused by Entomophaga maimaiga in Bulgaria and Serbia

Georgi Georgiev 1, Mara Tabaković-Tošić 2, Margarita Georgieva 1,*, Plamen Mirchev 1

1 Forest Research Institute - Bulgarian Academy of Sciences, 132 St. Kliment Ohridski Blvd., 1756 Sofia, Bulgaria 2 Institute of Forestry, Kneza Višeslava 3, 11030 Belgrade, Serbia

* Corresponding author: Margarita Georgieva; E-mail: [email protected]

Abstract: The impact of introduced fungal pathogen Entomophaga maimaiga on population density of Lymantria dispar (L.) was studied in Bulgaria and Serbia during the period 2009-2014. In many localities, strong mortality was observed not only during the larval development but also in the pupal stage of the host. The average annual gypsy moth mortality caused by E. maimaiga in Bulgaria varied between 66.5% and 86.8%, and in Serbia – between 62.8% and 98.8%. The pupal mortality in Bulgaria and Serbia varied between 11.7-33.1% and 0.4-6.3%, respectively. The analysis of the biological material showed that the number of dead pupae was considerably high, in spite of the established small amount of the pathogen’s azigospores. The number of azigospores in L. dispar dead pupae in three studied localities in Serbia varies strongly (12-30 per а view field), but the average values are very close (2.33-2.90).

Keywords: Lymantria dispar, Entomophaga maimaiga, host mortality, pupal stage, Bulgaria, Serbia.

1. Introduction

The gypsy moth, Lymantria dispar (Linnaeus, 1758) (Lepidoptera: Erebidae), is one of the most harmfulness insect pests in forest ecosystems of Europe, Asia, Japan and North Africa. Widely polyphagous, it is trophycally connected with more than 300 deciduous and coniferous tree and shrub species, mainly preferring oaks (Quercus spp.) and poplars (Populus spp.). In 1868, the gypsy moth was accidentally introduced from Europe to North America. In the new habitats, many introductions of parasitoids and pathogens of the pest have been conducted from its native range (Europe and Asia), including entomopathogenic fungus Entomophaga maimaiga Humber, Shimazu & Soper (Entomophthorales: Entomophthoraceae) (Hajek et al. 1995). Gypsy moth epizootics caused by E. maimaiga were first observed in 1989 in seven Northwestern states (Andreadis and Weseloh, 1990). Since that time, the fungus has been spread through most of the range of L. dispar in US and Canada, and is responsible for collapses of many local gypsy moth populations (Tobin and Hajek, 2012). In 1999, E. maimaiga was successfully introduced in Europe, in the region of Central Bulgaria (Pilarska et al. 2000). The first strong epizootics caused by the fungus were observed in 2005 in different areas of Northern and Southern Bulgaria (Pilarska et al. 2006). The fungus

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enlarged its range in many localities of the country through naturally spreading or new introductions (Georgiev et al. 2013). In 2011, E. maimaiga was established in Serbia (Tabaković- Tošić et al. 2012) and European part of Turkey (Georgiev et al., 2012). In 2012, E. maimaiga penetrated in Greece, Macedonia (Georgieva et al. 2013), Croatia (Hrašovec et al. 2013) and Romania (Netoiu et al. 2016), and in 2013 – in Bosnia and Herzegovina (Milotić et al. 2015), Hungary (Csóka et al. 2014) and Slovakia (Zubrik et al. 2014). E. maimaiga is a host specific, high virulent and very effective regulator of gypsy moth density and has great potential as a biological control agent. The life cycle of the pathogen is well synchronized with host larval development. It produces two types of spores: conidia and resting (azygospores). Conidia are formed mainly in young (early-instar) larvae, and mortality of the host occurs in tree crowns. Azygospores are formed in older (late-instar) larvae, and mortality occurs on tree stems. It is known that а part of infected gypsy moth larvae die in pupal stage (Hajek, 1999). In this paper we report the results of pupal mortality of L. dispar caused by E. maimaiga in host populations in Bulgaria and Serbia.

2. Material and Methods

The studies were conducted in 2011-2014 in 34 localities of L. dispar in which epizootics caused by E. maimaiga occurred in Bulgaria and Serbia. The main characteristics of studied localities and forest stands are pointed in the Tables 1 and 2.

Table 1. Main characteristics of studied areas in Bulgaria.

) Stand composition

Forest Geographical

Locality** a.s.l.

Age

Enterprise* coordinates Tree species % (year)

Altitude (m

SFE Gorna Asenovo vill. N43o17'41.7" 401 Quercus cerris 100 65 Oryahovitsa E26o04'50.0" SFE Govezhda Elovitsa vill. N43o19'51.0" 345 Carpinus orientalis 100 50 E23o00'14.8" Quercus cerris Single trees SFE Haskovo Spahievo vill. N41o52'51.4" 451 Quercus frainetto 100 55 E25o19'29.7" SFE Kirkovo Kremen vill. N41o17'08.0" 474 Quercus frainetto 70 60 E25o19'52.1" Quercus petraea 30 SFE Nova Zagora Sadievo vill. N42o31'46.9" 151 Quercus robur 100 50 E26o08'54.0" SHE Cherni Lom Slavyanovo vill. N43o17'05.4" 345 Quercus cerris 100 65 (Popovo) E26o08'03.1" SHE Staro Obzor vill. N42o47'32.8" 97 Quercus cerris 60 50 Oryahovo E27o52'32.8" Quercus frainetto 40 Ravna gora vill. N43o02'13.0" 40 Quercus cerris 60 50 E27o49'56.3" Quercus pubescens 40 Solnik vill. N42o54'12.9" 205 Quercus frainetto 60 80 E27o42'56.8" Quercus cerris 40 SFE Sredets Fakia vill. N42o13'25.7" 362 Quercus frainetto 70 40 E27o08'05.1" Quercus cerris 20 Fraxinus ornus 10 SFE Targovishte Dalgach vill. N43o12'57.9" 193 Quercus rubra 80 35 E26o42'28.7" Tilia platyphyllos 20 SFE Zvezdets Indzhe voyvoda vill. N42o13'17.7" 299 Quercus cerris 60 40 E27o27'00.4" Quercus frainetto 40 Zvezdets vill. N42o07'04.1" 336 Quercus petraea 90 75 E27o24'07.2" Quercus frainetto 10 Quercus cerris Single trees * SFE – State Forest Enterprise; SHE – State Hunting Enterprise; PE - Public Enterprise; FE – Forest Estate ** FA – Forest Administration

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Table 2. Main characteristics of studied areas in Serbia.

Stand composition

Forest Geographical Locality** Enterprise* coordinates Age

Tree species % (year)

Altitude (m a.s.l.)

PE Srbijašume Borački Gaj vill. N44°32' 120-170 Quercus cerris 80 65 FE Beograd (FA Lipovica) E20°21' Quercus frainetto 20 Diocese of Bogovađa N44°19' 140-190 Quercus cerris 60 82 Valjevo– E20°11' Carpinus betulus 30 Monastery Quercus frainetto 10 forests or Tilia argentea PE Srbijašume Istočna Boranja N44°22'41.28" 423 Fagus sylvatica 100 100 FE Boranja Loznica (FA Krupanj) E19°21'40.19" PE Srbijašume Brestovac vill. N44°02'21.24" 400 Quercus cerris 90 65 FE Timočke šume (FA Bor) E22°06'22.35" Boljevac Urovica vill. N44°24'51.38" 180 Quercus cerris 90 65 (FA Negotin) E22°21' 08.87" PE NP Đerdap Monte Miroč and N44°35'28" 560 Fagus sylvatica 100 90 Crni Vrh E22°16'49" (Reon Donji N44°34'13" 620 Fagus sylvatica 100 90 Milanovac) E21°53'47" PE Srbijašume Srndaljska reka N43°25'50.28" 593 Fagus sylvatica 100 90 FE Rasina Kruševac (FA Kruševac) E21°29'14.82" Žunjačko Batotske N43°19'56.31" 556 Fagus sylvatica 100 90 planine E21°11'04.35" (FA Brus) PE Srbijašume Mali Jastrebac N43°20'32.60" 537 Fagus sylvatica 100 80 FE Toplica (FA Prokuplje) E21°35'55.10" Kuršumlija Monte Javorac N43°18'04.98" 572 Fagus sylvatica 100 85 (FA Blace) E21°11'18.18" Dobri Do N42°53'10.89" 859 Fagus sylvatica 100 100 (FA Kuršumlija) E21°23'41.42" PE Šuma Goč Gračac N43°35'11.76" 559 Fagus sylvatica 100 100 Vrnjačka Banja E20°50'15.40" Monte Goč N43°33'03.36" 889 Fagus sylvatica 90 90 E20°55'16.03" PE Srbijašume Gledićke N43°46'49.07" 590 Fagus sylvatica 100 90 FE Stolovi Kraljevo planine E20°55'33.39" (FA Kraljevo) PE Srbijašume Monte N44°13'28" 659 Fagus sylvatica 100 80 FE Severni Kučaj Deli Jovan E22°15'09" Kučevo (FA Majdanpek) Lješnica vill. N44°29'58.00" 436 Fagus sylvatica 90 80 (FA Kučevo) E21°39'54.14" PE Srbijašume Knić–Pajsijević vill. N43°52'39.37" 537 Quercus cerris 50 65 FE Kragujevac (FA Kragujevac) E20°42'25.81" Quercus petraea 50 PE Srbijašume Monte Vučje N42°50'26.63" 546 Fagus sylvatica 95 90 FE Šuma Leskovac (FA Vučje) E21°53'19.99" Miroševce vill. N42°51'56" 285 Quercus cerris 90 65 (FA Leskovac) E21°50'21" PE Srbijašume Monte Rtanj N43°46'34" 623 Fagus sylvatica 100 90 FE Niš (FA Sokobanja) E21°53'36" * SFE – State Forest Enterprise; SHE – State Hunting Enterprise; PE - Public Enterprise; FE – Forest Estate ** FA – Forest Administration

In order to collect biological material, double-layered burlap bands (about 30 cm in width) were placed on 25 oak trees in each study site. The bands surrounded the tree trunks at a height of 1.3 m from the ground. Larvae of L. dispar were collected from the bands, which they use as resting sites, 2-3 times a month from early May to late July. Collected larvae were returned to the laboratory and reared in groups of 10-20 on fresh oak foliage in plastic boxes (15 × 10 × 8

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cm). The foliage was changed daily and dead larvae were separated individually in Petri dishes with water-saturated filter paper disc at 20°C for 4-5 days. The pupated specimens were also separated individually in Petri dishes for examination. The Petri dishes with dead L. dispar larvae and pupae were stored for a week at room temperature and then refrigerated at 5°C until microscopical evaluation. Microscopical analyses were made at magnification ×100, ×125 and ×400. Each cadaver of dead larva or pupa was dissected individually and observed under light microscope for the presence of conidia or azygospores of E. maimaiga. The number of E. maimaiga resting spores in dead pupae was calculated in three sites in Serbia on the base of three samples utilizing polyester micrometer cover slips (18×18 mm). Microscopic analyses were made with a microscope NU 2 at magnification ×125.

3. Results

The mortality of L. dispar caused by E. maimaiga in different localities in Bulgaria and Serbia varied between 42.1 and 100.0% (Tables 3 and 4).

Table 3. Lymantria dispar mortality in Bulgaria.

Studied larvae Mortality (%) Year Locality and pupae, N Larval Pupal Total 2009 Elovitsa 32 68.8 28.1 96.9 Kirkovo 73 52.1 31.5 83.6 Ravna gora 205 53.2 17.5 70.7 Sadievo 100 53.0 30.0 83.0 Spahievo 261 23.0 49.4 72.4 Zvezdets 146 58.9 29.5 88.4 2010 Asenovo 36 66.7 16.6 83.3 Elovitsa 46 41.3 15.2 56.5 Kirkovo 19 26.3 15.8 42.1 Ravna gora 148 64.9 9.4 74.3 Spahievo 42 59.5 9.5 69.0 Zvezdets 54 55.6 22.2 77.8 2011 Elovitsa 5 0.0 80.0 80.0 Dalgach 53 100.0 0.0 100.0 Fakia 273 57.5 23.8 81.3 Indzhe voyvoda 43 72.1 20.9 93.0 Obzor 108 28.7 49.1 77.8 Kirkovo 333 73.0 19.5 92.5 Slavyanovo 32 90.6 3.1 93.7 Solnik 89 92.1 2.3 94.4 Spahievo 147 52.4 15.6 68.0 Ravna gora 158 73.4 12.7 86.1 Zvezdets 434 44.7 45.9 90.6

In Bulgaria, the larval mortality of gypsy moth varied between 0% (Elovitsa, 2011) and 100% (Dalgach, 2011). On the other hand, in above mentioned localities the pupal mortality reached up highest and lowest values in this year – 80% and 0%, respectively. In Serbia, the larval mortality of L. dispar varied between 82.9% (Monte Javorac, 2013) and 100% (Gračac and Monte Deli Jovan, 2013; Monte Rogot, Miroševce and Monte Rtanj, 2014). The pulal mortality was relatively low and varied between 0% in six localities (Gračac, Monte Goč, Monte Deli Jovan, 2013; Monte Rogot, Miroševce and Monte Rtanj, 2014) and 6.9% (Beograd, 2011) (Table 4). The average annual gypsy moth mortality caused by E. maimaiga in three studied years in Bulgaria varied between 66.5% (2010) and 86.8% (2011) (Figure 1). The larval and pupal mortality varied between 45.0-60.5% and 11.7-33.1%, respectively.

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Table 4. Lymantria dispar mortality in Serbia.

Studied larvae Mortality (%) Year Locality and pupae, N Larval Pupal Total 2011 Beograd 532 85.0 6.9 91.9 Valjevo 321 92.5 5.3 97.8 2012 Negotin 416 94.0 4.1 98.1 Monte Miroč and Crni Vrh 536 91.8 5.0 96.8 2013 Srndaljska reka 721 96.8 2.1 98.9 Žunjačko Batotske planine 1347 88.7 8.4 97.1 Mali Jastrebac 271 94.5 2.2 96.7 Monte Javorac 532 82.9 3.2 86.1 Dobri Do 931 97.8 2.2 100.0 Gračac 801 100.0 0.0 100.0 Monte Goč 395 96.2 0.0 96.2 Gledićke planine 877 86.7 9.7 96.4 Lješnica 222 90.5 4.9 95.4 Monte Deli Jovan 109 100.0 0.0 100.0 Majdanpek 434 94.5 2.3 96.8 Monte Vučje 298 98.0 2.0 100.0 Istočna Boranja 211 94.1 5.9 100.0 2014 Monte Rogot 143 100.0 0.0 100.0 Brestovac 117 93.2 5.1 98.3 Miroševce 213 100.0 0.0 100.0 Monte Rtanj 831 100.0 0.0 100.0

Larval mortality Pupal mortality Total mortality 100 86.8 78.1 80 66.5 60.5 60 54.8 45

40 33.1

Percentage 26.3

20 11.7

0 2009 2010 2011

Figure 1. Average annual mortality of Lymantria dispar caused by Entomophaga maimaiga in Bulgaria.

Larval mortality Pupal mortality Total mortality 99.4 99.8 100 97.4 97 92.8 93

80 62.8 60 56.5

40 Percentage

20 6.3 4.6 4 0.4 0 2011 2012 2013 2014

Figure 2. Average annual mortality of Lymantria dispar caused by Entomophaga maimaiga in Serbia

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Figure 3. Density of Entomophaga maimaiga azygospores extracted from Lymantria dispar dead pupae in 2013 in Serbia: 1 - Srndaljska reka (N=83); 2 - Mali Jastrebac (N=26); 3 - Monte Vučje (N=15)

In Serbia, the average annual mortality varied between 62.8% (2011) and 98.8% (2014) (Figure 2). The larval mortality in four studied years was 56.5-99.4%, and pupal one – 0.4-6.3%. The number of azigospores in L. dispar dead pupae in three studied localities in Serbia varies strongly (12-30 per а view field), but the average values are very close (2.33-2.90 per а view field) (Figure 3).

4. Discussion

It is noteworthy, that the results of L. dispar mortality caused by E. maimaiga in Bulgaria and Serbia differ significantly. For example, lower total mortality rates (66.5-86.8%) were reported in Bulgaria, compared to Serbia (62.8-99.8%). These differences are even greater for pupal mortality – 11.7-33.1% in Bulgaria and only 0.4-6.3% in Serbia. Undoubtedly, the reason for this phenomenon is different methodological approach to data reporting – in this paper, there is no data about many field epizootics in gypsy moth populations in Bulgaria, where the larval mortality rates were, as a rule 100%, and the host could not reach the pupal stage. These include mass epizootics in 2005 (Pilarska et al. 2006), epizootics as a result of artificially introductions in 2008-2011 (Georgiev et al. 2013) and the spontaneous epizootics that occurred in Kirkovo region in 2013-2014 (Georgiev et al. 2014). The use of these data would, on the one hand, lead to an increase in the values of larval and total mortality of gypsy moth, and a reduction in pupal mortality rates, on the other hand. It is well known that the environmental conditions are of great importance for the spread of E. maimaiga and and its impact on the host. Production and density of conidia are higher in the areas with higher relative humidity, often occurred after the rain (Hajek et al. 1990; Hajek et al. 1999). Spore production and release was higher at 95-100% relative humidity, and only limited spore production was seen at 50-70% relative humidity. Conidia required also free water for germination (Hajek et al. 1990). Humid spring time in late April and May is positively associated with infections by E. maimaiga. Under favorable conditions, the conidial infections affect early- instar gypsy moth larvae and cause a massive mortality prior to causing defoliation on the host plants. In this case, the host died long time before pupating.

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Infection with germ conidia of resting spores can also occur later when late-instar gypsy moth larvae descend the host trees to rest during the day on tree trunks or on forest litter. Epizootics usually occur near the end of larval development, where tree trunks and branches are covered with dead larvae. It could be noted that the production of azigospores is also positively associated with temperature and moisture level (Shimazu, 1987). Under unfavorable conditions and low extensiveness of fungal infection, pupation of infected late-instar L. dispar larvae should be expected with subsequent mortality in the pupal stage. In conclusion, the pupal mortality of L. dispar caused by the fungal pathogen is of great importance for health status assessment of pest populations. E. maimaiga can be regarded as a good alternative to the use of chemical and bacterial insecticides for pest control, which is of great importance for the quality of human environment and biodiversity conservation in forest ecosystems.

5. References

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